Abstract
Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 °C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 ± 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 ± 0.22 × 1012 particle/mL and 8.3 ± 1.7 × 1010 particle/µg, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.
Highlights
Exosomes are a subtype of extracellular vesicles (EV) ranging from 50-200 nm in diameter, secreted by various cell types such as immune cells[1,2], cancer cells[3,4,5,6] and stem cells[7]
Exosome yield from the HEK-293 cells, analyzed using the Nanoparticle Tracking Analysis (NTA) instrument, was 6.99 ± 0.22 x 1012 p/mL from ~24 mL of conditioned medium (CM)
Obtaining a decent exosome yield from cultured cells, which are enough for several rounds of in vitro or in vivo studies, is still a challenge
Summary
Exosomes are a subtype of extracellular vesicles (EV) ranging from 50-200 nm in diameter, secreted by various cell types such as immune cells[1,2], cancer cells[3,4,5,6] and stem cells[7]. Various small molecules that serve as anti-cancer and anti-inflammatory drugs have been demonstrated to be successfully loaded into exosomes and delivered to target cells[18,19,20,21,22,23,24,25,26,27]. Various methods have been reported to successfully isolate exosomes from either cell culture or physiological fluids. We propose an exosome isolation protocol using density-based ultracentrifugation onto just a single 25% (w/w) sucrose cushion prepared in deuterium oxide, rather than a sucrose density gradient This is a cost-effective method that circumvents the laborious density gradient preparation and allows processing of large volumes of starting material, yet results in intact exosomes of high yield and purity suitable for subsequent loading with siRNA. For the 2nd and subsequent harvest, the CM is kept for exosome isolation
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